Gentianopsis metabolites and bioactivity: HPLC-PDA-ESI-tQ-MS/MS profiles, HPLC-UV quantification of Gentianopsis komarovii and Gentianopsis stricta, and choleretic potential

Abstract Gentianopsis is a small gentianaceous genus with a known ethnopharmacological focus as hepatoprotectors containing two underestimated species that are scientifically unexplored: Gentianopsis komarovii (Grossh.) Toyok., which is typical of the Far East, and Gentianopsis stricta (Klotzsch) Ikonn., which is grown in Central Asia. Application of the HPLC-PDA-ESI-tQ-MS/MS technique led to the identification of 28 compounds, such as iridoid glycosides, flavones and xanthones, with loganic acid, sweroside, loganin, secologanin, isoorientin-7-O-glucoside, luteolin-7-O-gentiobioside, chrysoeriol-7-O-glucoside and acacetin-7-O-glucoside being found in the genus for the first time. The extracts of G. komarovii and G. stricta demonstrated choleretic potential, strengthening the bile flow and the total content of bile acids, bilirubin and cholesterol in the bile. The most pronounced effects were observed for luteolin-7-O-glucoside and gentiabavaroside (gentiacaulein-1-O-primveroside), establishing them as the principle choleretics of both herbs. Based on the results, G. komarovii, G. stricta and some phenolic metabolites are prospective new choleretic drugs. Graphical Abstract


Introduction
Since the outbreak of the novel coronavirus infection (COVID-19) pandemic in 2019, scientists and clinicians around the world have been constrained by the lack of effective etiological treatments.Data on the effectiveness of some antiviral drugs in combination with antibiotic therapy made it possible to recommend them for the treatment of patients with COVID-19 (Vitiello et al. 2021); however, antiviral drugs provoke the development of undesirable reactions in various systems and organs of the body, including the liver, which require correction in order to continue treatment (Yu et al. 2021).The described cases of adverse reactions of the liver are characterized mainly by impaired functional activity; therefore, hepatoprotective drugs are required, to increase the resistance of the liver to the damaging effects of various pathogens and help restore the functional activity of the hepatocytes (Smok et al. 2020).One of the promising areas of symptomatic therapy for liver damage in COVID-19 is the use of phytochemicals that have a positive hepatoprotective effect with minimal side effects (Vargas-Mendoza et al. 2021).Species of the Gentianaceae family are among the effective hepatoprotectors of plant origin, including Gentiana (Jiang et al. 2021), Gentianella (Li et al. 2010) and Swertia (Li et al. 2017).
Gentianopsis is a small genus of annual and biennial herbs of the Gentianaceae family, which includes about 20 species and is distributed mainly in Asia, Europe and North America (Ding et al. 2011).Few studies that have investigated the metabolites of twenty Gentianopsis plants found carbohydrates, flavonoids, xanthones, iridoids and triterpenes totalling 53 compounds (Supplementary material, Tables S1 and S2).The major substances were c-pyrones as glucoside derivatives of apigenin, luteolin, diosmetin, gentiakochianin, gentiacaulein and decussatin.The extracts of Gentianopsis and the pure compounds were found to be hepatoprotective, antiproliferative, cytotoxic, antidiarrheal and antimycobacterial drugs (Supplementary material, Table S3).As part of our ongoing study of gentianaceous plants (Olennikov et al. 2019;Olennikov andChirikova 2021a, 2021b), we selected two Gentianopsis species that have satisfactory natural reserves and no current level of knowledge.One of them was Gentianopsis komarovii (Grossh.)Toyok., the usual plants in Far East grasslands, and the other was Gentianopsis stricta (Klotzsch) Ikonn.{G.vvedenskyi (Grossh.)Pissjauk.},which grows in swampy places and wet lawns of the alpine belt of Central Asia.Both plants have similar ethnopharmacological applications as hepatoprotective and antiinflammatory remedies (Budantsev 2011;Akobirshoeva 2012).In the present work, we studied the metabolic profiles and quantitative patterns of the two Gentianopsis species using high-performance liquid chromatography with photodiode array and electrospray ionization triple quadrupole mass spectrometric detection technique (HPLC-PDA-ESI-tQ-MS/MS) and investigated the effects of the extracts and pure compounds on the bile flow and composition in rats through duodenal administration.

2: Results and discussion
Twenty-eight compounds were found in the organs of the two Gentianopsis species after HPLC-PDA-ESI-tQ-MS/MS profiling and identification procedures, including a comparison of retention times, and ultraviolet and mass-spectrometric data with known standard compounds (Supplementary material, Tables S4 and S6).There were five iridoid glycosides, ten xanthone aglycones, Cand O-glycosides, and thirteen flavonoid aglycones, C-, Oand C,O-glycosides (Supplementary material, Figures S1-S7).The iridoids were the known Gentianopsis compound swertiamarin (Cui et al. 2013) and four compounds newly found in the genus: loganic acid, sweroside, loganin and secologanin, usual for other Gentianaceous species (Venditti et al. 2013).Mangiferin was the only C-glycosylxanthone detected in both species, together with the known compounds gentiacaulein, gentiakochianin and decussatin, and their O-glucosides and Oprimverosides (Massias et al. 1982).The flavonoids were flavone derivatives of apigenin, luteolin, diosmetin and acacetin, of which isoorientin-7-O-glucoside, luteolin-7-Ogentiobioside, chrysoeriol-7-O-glucoside and acacetin-7-O-glucoside were found in Gentianopsis for the first time.The study of the compound distribution in organs of G. komarovii and G. stricta showed an accumulation of iridoid glycosides, mangiferin and flavones in the leaves and xanthone-O-glycosides in the flowers (Supplementary material, Tables S6 and S7 and Figures S8 and S9).The total content of iridoid glycoside, xanthones and flavones in the organs was 0.27-6.12mg/g, 6.98-52.47mg/g and 0.36-44.41mg/g, respectively.The known chemosystematic conclusions (Massias et al. 1982) related to the distribution of phenolic compounds inside the Gentianopsis genus indicate that the 1,3,7,8-substituted xanthones are the chemical markers found in all species, and the new data confirm this early opinion.
To investigate the choleretic potential of G. komarovii and G. stricta, we prepared methanolic extracts of the herbs and demonstrated the close profiles of the herbs and final extracts using HPLC-PDA-ESI-tQ-MS/MS data (Supplementary material, Table S8).The bioactivity was studied using acclimatized and anesthetized Wistar rats, following cannulation and intraduodenal application of the extracts in doses of 25, 50 and 100 mg/kg.The positive control was dehydrocholic acid (DHCA, 20 mg/kg) (Wang et al. 2016), and saline was used as a negative control.The DHCA showed the maximum increase of bile flow after 30 min (12 mg/min/100 g), as opposed to the saline group, with 8.2 mg/min/100 g bile flow (Supplementary material, Figure S10).The total bile acid content of the bile sample in the DHCA group was comparable with the saline group (1563 mg/100 g vs. 1463 mg/100 g, respectively), while the values of total bilirubin (42.18 mg/100 g vs. 27.19mg/100 g; p < 0.001) and total cholesterol (31.16 mg/ 100 g vs. 26.44 mg/100 g; p < 0.001) were higher (Supplementary material, Table S9).These results point to the insignificant influence of DHCA on the total bile acid basal excretion.
Application of the Gentianopsis extracts gave an increased bile flow in groups with doses of 50 mg/kg (9.92-10.23 mg/min/100 g) and 100 mg/kg (11.24-11.60mg/min/ 100 g), with similar efficacy of both extracts (Supplementary material, Figure S10).The total bile acid content in the experimental groups with 100 mg/kg Gentianopsis extracts were higher than the saline group (2039-2132 mg/100 g; p < 0.001), as were the levels of total bilirubin and total cholesterol, which were 56.14-58.22mg/100 g (p < 0.001) and 32.63-33.90mg/100 g (p < 0.001), respectively (Supplementary material, Table S9).This highlights the positive choleretic effects of G. komarovii and G. stricta extracts on basal bile secretion.
Finally, we studied the effects of three pure compounds on the bile flow and bile composition after duodenal administration.The compounds selected were the dominant phytochemicals of the iridoid glycoside group (secologanin), the flavone group (luteolin-7-O-glucoside) and the xanthone group (gentiabavaroside), which were administered in a single dose of 20 mg/kg (as with DHCA).The highest increase of bile flow was found after 30 min of administration in the luteolin-7-O-glucoside group (þ72% vs. saline group; p < 0.001), followed by the DHCA group (þ45% vs. saline group; p < 0.001) and the gentiabavaroside group (þ33% vs. saline group; p < 0.001).The efficacy of the secologanin group on the bile flow was insignificant.The application of luteolin-7-O-glucoside showed the greatest impact on the bile composition, increasing the total bile acid content (þ96% vs. saline group; p < 0.001), total bilirubin content (þ203% vs. saline group; p < 0.001) and total cholesterol content (þ46% vs. saline group; p < 0.001).The efficacy of gentiabavaroside was slightly less than luteolin-7-O-glucoside, and secologanin was the least effective.Thus, the flavones and the xanthones are the main choleretic metabolites of G. komarovii and G. stricta extracts.
This study demonstrates that the extracts of two Gentianopsis species (G.komarovii and G. stricta) with an ethnopharmacological background as choleretics displayed an increased bile flow and an increase in total bile acids, total bilirubin content and total cholesterol content.Gentianopsis barbata extract has previously shown hepatoprotective and anticholestatic effects (Nikolaev et al. 2001).Some plant flavonoids are known choleretic agents, such as luteolin (Gebhardt 2005), liquiritigenin (Kim et al. 2009), quercetin and silymarin (Negi et al. 2008), while the hepatoprotection of xanthones is, at present, poorly understood (Pinto et al. 2005).Toxicity data of flavones and xanthones has also shown their safety (Galati and O'Brien 2004;Pinto et al. 2005).Even though additional pharmacological studies are needed for luteolin-7-O-glucoside and gentiabavaroside, we have concluded that both compounds and both parent plants are prospective new choleretic drugs.This is particularly true in the case of COVID-19 patients who are overloaded with hepatotoxic drugs and need harmless liver protection.Some phytochemical hepatoprotectors like curcumin, silymarin and sulphoraphane, among others, have already been shown to have the functional utility to treat SARS-CoV-2 infection (Vargas-Mendoza et al. 2021).Considering their good choleretic potential, G. komarovii and G. stricta extracts and their two active metabolites, luteolin-7-O-glucoside and gentiabavaroside, are new potential candidates for effective natural choleretics or functional supplements.

3: Conclusions
The present study demonstrates that two underestimated Gentianopsis species, G. komarovii and G. stricta, have similar chemical profiles, with a dominance of flavones, xanthones and iridoid glycosides.The ethnopharmacological data on the choleretic efficiency of these plants has been proven by the experimental evidence, and two compounds, luteolin-7-O-glucoside, and gentiabavaroside, were found to be the most effective choleretics.Our findings may provide insight into the role of gentianaceous plants and their metabolites in the treatment of liver diseases and improve our knowledge of new hepatoprotectors.